CN111363691B - Paenibacillus polymyxa and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention provides a Paenibacillus polymyxa strain and application thereof, wherein the strain is named as Paenibacillus polymyxa HY31, and the preservation number is CCTCC No: M20191102. The bacterial strain has strong inhibition effect on fusarium oxysporum cucumber specialization, fusarium oxysporum tomato specialization, fusarium oxysporum pepper specialization, fusarium oxysporum wilting specialization and other plant fusarium wilt pathogenic bacteria, and the bacterial agent prepared by the bacterial strain can prevent the fusarium wilt and promote the growth of vegetables when being applied to the vegetables.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to paenibacillus polymyxa and application thereof.
Background
Blight, also known as epidemic disease, is a worldwide soil-borne disease caused by fungi or bacteria, which has a wide distribution range, many types of strains, difficulty in control, and serious impact on crop yield and quality. Sudden onset of disease, symptoms include severe spotting, wilting or death of leaves, flowers, fruits, stems or whole plants. Most important commercial crops such as tomatoes, cucumbers, cotton and the like are infected by one or more epidemic diseases. Blight can affect flowers, leaves, bud bags, seedlings, twigs, stems and tips. The blight is a main epidemic disease in China, and due to the fact that the number of pathogenic bacteria transmission media is large, active and passive transmission can be conducted through natural flowing, transporting and moving of germ-carrying crop seedlings, diseased plant residues, farming tools, soil, running water and the like; in addition, with the adjustment of agricultural industrial structure, the facility agriculture mainly comprising greenhouses and greenhouses is developed rapidly, particularly in vegetable planting, continuous cropping of crops is increasingly serious, and some vegetable planting bases, planting parks and the like can be used for continuously planting the same kind of vegetables for several years or even more than ten years; the chlamydospores of pathogenic bacteria of the blight have long survival time in soil, and the blight in vegetables is seriously attacked and difficult to be radically cured due to the combination of a plurality of reasons.
At present, three measures such as breeding disease-resistant varieties, agricultural measures, chemical control and the like are generally adopted for preventing and treating the vegetable blight, but the three methods have problems, such as low disease resistance of the varieties and easy loss of resistance; the chemical control agent has poor later-period control effect, pathogenic bacteria are easy to generate drug resistance, and the environment negative effect is obvious; agricultural measures are easily limited by regional conditions and difficult to popularize in a large area, and the like. In recent years, with the continuous deepening of sustainable and ecological agriculture concepts, biological control is more and more emphasized by people, and the method for controlling bacteria by bacteria has obvious effect and no adverse effect.
The Paenibacillus polymyxa exists in a spore form, has good temperature resistance and stable quality, treats bacteria with bacteria and has lasting drug effect; the pesticide and fertilizer can not only be the most ideal pesticide for preventing and treating soil-borne diseases such as bacterial wilt, fusarium wilt, root rot, soft rot and the like, but also have the obvious effects of promoting growth, increasing yield and improving quality; the microbial preparation has no chemical injury and residue, and is a preferred microbial preparation for producing pollution-free, green and organic vegetables; has obvious prevention effect on vegetable blight. The united states Environmental Protection Agency (EPA) has listed it as one of the commercially available microbial species. The paenibacillus polymyxa kills and controls pathogenic bacteria through the action mode that antibacterial substances generated by the paenibacillus polymyxa compete with sites and resistance is induced, thereby achieving the purpose of preventing and treating diseases.
Disclosure of Invention
In view of the above, the invention provides a Paenibacillus polymyxa and application thereof, wherein the Paenibacillus polymyxa is selected from Wuhan Jiangxiao and Xinzhou vegetable bases and collected from continuous cropping land and the periphery of a vegetable root system with serious diseases, so that the Paenibacillus polymyxa not only has a good control effect on the blight of vegetables, but also is a green, environment-friendly and effective vegetable blight disease control way; but also has obvious effects of promoting growth and increasing yield of vegetables.
The technical scheme of the invention is realized as follows:
in a first aspect, the invention provides a strain of Paenibacillus polymyxa, which is classified and named as Paenibacillus polymyxa HY31 and is preserved in China Center for Type Culture Collection (CCTCC) in 12 months and 25 days in 2019 at the address of Wuhan university in Wuhan, China, zip code 430072 with the preservation number of M20191102.
In a second aspect, the invention provides an application of paenibacillus polymyxa HY31 in antagonizing fusarium wilt pathogens, including an application of antagonizing fusarium oxysporum cucumber specialization, fusarium oxysporum tomato specialization, fusarium oxysporum pepper specialization, fusarium oxysporum wilting specialization and other pathogens.
In a third aspect, the invention provides application of paenibacillus polymyxa HY31 in prevention and treatment of vegetable blight diseases.
In a fourth aspect, the invention provides a microbial agent, which comprises paenibacillus polymyxa HY 31.
Based on the above technical scheme, the preferable viable bacteria concentration of the paenibacillus polymyxa HY31 is at least 10 9 cfu/mL。
In a fifth aspect, the invention provides the use of the microbial agent of the fourth aspect in the prevention and treatment of vegetable blight disease.
Compared with the prior art, the paenibacillus polymyxa and the application thereof have the following beneficial effects:
(1) the invention screens the paenibacillus polymyxa HY31 from the periphery of the root system of the vegetable with serious disease in the continuous cropping soil, has better inhibition effect on fusarium oxysporum cucumber specialization, fusarium oxysporum tomato specialization, fusarium oxysporum pepper specialization, fusarium oxysporum wilting specialization and other fusarium wilt pathogenic bacteria, and avoids the problems of chemical prevention and control medicament abuse, poor later-period prevention effect and obvious negative effect on the environment;
(2) the microbial agent prepared from the screened paenibacillus polymyxa HY31 is applied to vegetables, so that the blight of the vegetables can be well prevented, and the growth of the vegetables can be promoted.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram showing the effect of a fermentation broth of Paenibacillus polymyxa HY31 on the inhibition of the specialization type of Fusarium oxysporum cucumber;
FIG. 2 is a diagram showing the control effect of the fermentation broth of Paenibacillus polymyxa HY31 on fusarium wilt of cucumber.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The media formulations referred to in the following examples:
yeast extract solid medium: 1.0 percent of sucrose, 0.45 percent of yeast extract and KH 2 PO 4 0.05％，MgSO 4 ·7H 2 0.05% of O, 0.05% of NaCl, 1.5% of agar powder and the balance of distilled water, wherein the pH value is 7.2.
Nutrient broth culture medium: 10g of peptone, 3g of beef extract, 5g of sodium chloride, and distilled water to reach the constant volume of 1000mL and the pH value of 7.4.
PDA solid medium: 200g of potato, 20g of glucose and 15g of agar, and adding distilled water to reach the constant volume of 1000 mL.
The above medium was sterilized with high-pressure steam at 121 ℃ for 20min before use.
Example 1 screening and identification of Paenibacillus polymyxa
S1 test Material
Soil samples are from vegetable planting bases in Wuhan Jiangxiao and New Zhou and are collected from continuous cropping lands and around vegetable roots with serious diseases.
The pathogenic bacteria are represented by fusarium oxysporum cucumber specialization, fusarium oxysporum tomato specialization, fusarium oxysporum pepper specialization and fusarium oxysporum wilting specialization, and are gifted by the university of china agriculture plant science and technology academy.
S2 preliminary screening of bacterial strain
And (3) coating a soil sample on a yeast extract culture medium plate by a gradient dilution method, and culturing in an incubator at 35 ℃. After 3 days of culture, selecting single colonies with different morphological characteristics, carrying out streak culture in three regions on a yeast extract culture medium, repeatedly separating and purifying for 3 times, and finally carrying out co-screening to obtain 36 strains with different morphologies, wherein the strains are respectively numbered HY01-HY 36.
S3 bacterial inhibition experiment rescreening
Adopting a bacterial plaque inhibition method, punching four small holes with the diameter of 10mm on the peripheral diagonal line of the center of a blank yeast extract culture medium plate, removing the culture medium, and respectively inoculating 4 different pathogenic bacteria bacterial plaques. Respectively inoculating and separating bacteria HY01-HY36 in the center of the culture medium plate, and culturing at 35 deg.C. The growth conditions of the isolate and the pathogenic bacteria and the antagonistic inhibition effect are recorded day by day. After measuring the diameter of the inhibition zone by using a bacterial plaque inhibition method, 4 strains with larger inhibition zones are screened out, and the numbers are HY18, HY21, HY31 and HY36 respectively. Then, the 4 strains are subjected to bacteriostatic experiments, 3 repeats are set for each strain, the flat plate is placed at 35 ℃ for culture, and the diameter of a bacteriostatic zone is determined after 72 hours. The results show that the HY31 strain has the largest diameter of inhibition zone for 4 pathogenic bacteria, so that the HY31 strain screened by the method has the best inhibition effect.
S4, identifying strains
After primary screening and secondary screening, the obtained HY31 strain is sent to microorganism product quality supervision and inspection testing center (Wuhan) of Ministry of agriculture for determination of physiological and biochemical indexes. The HY31 strain is positive by gram staining microscopy, has rod-shaped cells, spores, ellipses, growing in the spores to the terminal, and expanded blastocysts. On the nutrient agar plate, the bacterial colony is small, round and grey white, the surface is wet, slightly convex, flat and translucent, and the edge is irregular. The results of the physiological and biochemical characterization are shown in Table 1.
TABLE 1 physiological and biochemical characteristics of HY31 Strain
The HY31 strain is identified by a molecular biology method, the 16S rDNA sequence of the strain is measured, as shown in SEQ ID NO.1 in a sequence table, BLAST comparison is carried out in a GenBank nucleic acid database, a phylogenetic evolution tree of the HY31 strain is constructed, and the homology of the 16S rDNA sequence of the HY31 strain and a login GU369972.1(Paenibacillus polymyxa RCP6) sequence reaches 99.03%. Through the identification results of physiological and biochemical characteristics, phylogenetic evolution position and the like of the HY31 strain, the HY31 strain is finally identified as the Paenibacillus polymyxa HY31(Paenibacillus polymyxa HY 31).
The HY31 strain has been collected in China Center for Type Culture Collection (CCTCC) at 12 months and 25 days in 2019, with address of Wuhan university, Loudan 430072, Wuchang Loudan mountain, Wuhan, Hubei, and Collection number of M20191102.
Example 2 bacteriostatic experiment of Paenibacillus polymyxa HY31
Preparation of S1 and Paenibacillus polymyxa HY31 bacterial liquid
Paenibacillus polymyxa HY31 was inoculated into nutrient broth and shake-cultured at 37 ℃ and 200rpm for 12 h.
S2 preparation of pathogenic bacteria block
Inoculating 4 kinds of pathogenic bacteria of Fusarium oxysporum cucumber specialization type, Fusarium oxysporum tomato specialization type, Fusarium oxysporum pepper specialization type and Fusarium oxysporum wilting specialization type to the center of PDA solid culture medium, and culturing at 28 deg.C for 7 d.
S3 bacteriostatic test
The 4 pathogenic bacteria blocks (phi 10mm) are respectively placed in the center of a PDA solid culture medium plate (phi 90mm), and after a bacillus polymyxa HY31 bacterial liquid is dipped on the edge of a sterile blank plate with the diameter of 60mm, annular inoculation is carried out on the periphery of the pathogenic bacteria blocks in the PDA solid culture medium plate. The blank control group was inoculated with sterile water in a circular manner. Placing the inoculated PDA solid culture dish in an incubator at 28 ℃, measuring the diameters of pathogenic bacteria when culturing for 72h, 96h, 120h and 144h respectively, and calculating the bacteriostasis rate by using the following formula, wherein the specific result is shown in Table 2.
The bacteriostasis rate (%) is (diameter of the block of pathogenic bacteria in blank group-diameter of the block of pathogenic bacteria in treatment group)/diameter of the block of pathogenic bacteria in blank group x 100%
TABLE 2 bacteriostatic effect of Paenibacillus polymyxa HY31 on pathogenic bacteria
As can be seen from Table 2, the screened paenibacillus polymyxa HY31 can obviously inhibit the propagation of fusarium oxysporum cucumber specialization, fusarium oxysporum tomato specialization, fusarium oxysporum pepper specialization and fusarium oxysporum wilting specialization, the inhibition rate basically reaches the peak value when the cultivation lasts for 120 hours, the inhibition rates are all more than 57%, and the effect is obvious.
Example 3 test for controlling vegetable diseases with Paenibacillus polymyxa HY31
S1 inhibiting effect of paenibacillus polymyxa HY31 fermentation liquor on fusarium oxysporum cucumber specialization
Inoculating Paenibacillus polymyxa HY31 in nutrient broth liquid culture medium, shake culturing at 37 deg.C and 200rpm for 48 hr until the concentration of the fermentation liquid reaches10 9 cfu/mL。
Three experiments are set for researching the inhibition effect of the paenibacillus polymyxa HY31 fermentation liquor on the fusarium oxysporum cucumber specialization type: control, low dose, and high dose groups. Firstly, preparing 9 PDA solid culture medium flat plates, coating pathogenic bacteria on the flat plates, arranging 3 flat plates in each group, taking a sterile oxford cup in the center of the PDA flat plates, respectively adding 100 mu L of sterile water, 100 mu L of 5-time diluted paenibacillus polymyxa HY31 fermentation liquor and 100 mu L of paenibacillus polymyxa HY31 fermentation liquor into the oxford cup in a control group, a low-dose group and a high-dose group, then, arranging the flat plates in an incubator at 28 ℃ for culture, and observing the result after 72 hours. As shown in figure 1, the high-dose group and the low-dose group both obviously show bacteriostatic zones with different sizes, while the control group has no bacteriostatic zone, so that the bacillus polymyxa HY31 fermentation liquid has obvious inhibitory action on fusarium oxysporum cucumber specialization type, and the higher the concentration of the fermentation liquid is, the more obvious the inhibitory action is.
S2, Paenibacillus polymyxa HY31 for preventing and treating blight
According to the invention, the research on the control effect of paenibacillus polymyxa HY31 on blight is carried out on the milk cucumber in field tests, the field tests are divided into a control group and a test group, the test group uses the paenibacillus polymyxa HY31 fermentation liquor of the research, and the control group uses the same dose of clear water. The method is applied on the third day after transplanting and field planting of the gherkin, the fermentation liquor of paenibacillus polymyxa HY31 is diluted by 100 times, 100mL of the fermentation liquor is added to the roots of the gherkin seedlings, 300mL of the fermentation liquor is sprayed on the front and back sides of the gherkin leaves at the same time, and the fermentation liquor is sprayed for 1 time every 7 days and is continuously used for 3 times. As shown in FIG. 2, the test results of the cucumber plants were obtained for 60 days. As can be seen from the figure, the Ruhuang melon in the control group is obviously suffered from blight, the leaves are yellow, a plurality of bacterial plaques are contained, and the fruits are tiny; the test group added with the fermentation liquid of the paenibacillus polymyxa HY31 has no disease, the plants grow happy and the fruits are thick. The test result also proves the control effect of the paenibacillus polymyxa HY31 on the vegetable blight, and the research also finds that the paenibacillus polymyxa HY31 has a growth promoting effect on the vegetables.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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<120> Paenibacillus polymyxa and application thereof
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gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc gacctctggg ctgtaactga 720
cgctgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780
aaacgatgaa tgctaggtgt taggggtttc gatacccttg gtgccgaagt taacacatta 840
agcattccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg acggggaccc 900
gcacaagcag tggagtatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960
gacatccctc tgaccggtct agagatagac ctttccttcg ggacagagga gacaggtggt 1020
gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
ccttatgctt agttgccagc aggtcaagct gggcactcta agcagactgc cggtgacaaa 1140
ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1200
tactacaatg gccggtacaa cgggaagcga aggcgcgatg tggagccaat cctagaaaag 1260
ccggtctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa ttgctagtaa 1320
tcgcggatca gcatgccgcg gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca 1380
ccacgagagt ttacaacacc cgaaagtcgg tggagggaaa ccgcaaagga gccctgcccg 1440
ccct 1444
Claims (5)
1. Paenibacillus polymyxa, which is characterized in that: the Paenibacillus polymyxa is used for antagonizing fusarium wilt pathogenic bacteria, and is Paenibacillus polymyxa HY31 with the preservation number of CCTCC No: M20191102; the pathogenic bacteria of the fusarium wilt are fusarium oxysporum cucumber specialization type, fusarium oxysporum tomato specialization type, fusarium oxysporum pepper specialization type and fusarium oxysporum wilting specialization type.
2. The use of the Paenibacillus polymyxa of claim 1 for the control of vegetable blight disease.
3. A microbial inoculant characterized by: the microbial agent comprises the Paenibacillus polymyxa of claim 1.
4. The microbial inoculant according to claim 3, wherein: the viable bacteria concentration of the Paenibacillus polymyxa is at least 10 9 cfu/mL。
5. The use of the microbial inoculum of claim 3 in the control of vegetable blight disease.
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Denomination of invention: A Bacillus polymyxoides strain and its application Effective date of registration: 20230308 Granted publication date: 20220830 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: WUHAN HEYUAN GREEN ORGANISM Co.,Ltd. Registration number: Y2023420000094 |
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Denomination of invention: A Polymyxoid Bacillus Strain and Its Application Granted publication date: 20220830 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: WUHAN HEYUAN GREEN ORGANISM Co.,Ltd. Registration number: Y2024980008736 |